Molecular Cloning/Extraction of DNA

Large scale isolation of genomic DNA from cells grown as monolayers in tissue culture flasks or tissue samples (e.g. liver) from sacrificed animals. Briefly, to 10 ml of extraction buffer (10 mM Tris.Cl pH 8.0, 0.1M EDTA pH 8.0, 20 g/ml pancreatic RNase, 0.5% SDS) in a polypropylene tube 2.5 x 107 PCC4 cells (trypsinised and suspended in TE pH 8.0) or 100 mg tissue (frozen in liquid nitrogen and powdered by crushing) was added. The contents were mixed completely by inversion and incubated for 1 hr at 37C. To the lysed cells 1 mg of proteinase K was added at a final concentration of 100 g/ml (from a 20 mg/ml stock) mixed gently by inversion and incubated at 37C for 3-5 hr or overnight with frequent mixing during incubations. After proteinase K digestion, a viscous solution completely free of debris was observed. The solution was cooled, an equal volume of Tris-saturated phenol was added, mixed on a roto-torque to mix the two phases and then centrifuged at 10,000g for 5 min at room temperature. The upper clear aqueous viscous phase was transferred using a 5 ml pipette with a cut tip. The aqueous phase was further subjected to two rounds of phenol-chloroform and one round of chloroform extraction. The aqueous phase was then transferred to a clean polypropylene tube, 0.1 volume of 3 M sodium acetate (pH 4.2) and 2 volumes of absolute ethanol were added at room temperature and the contents mixed. The genomic DNA precipitate was spooled using a 1 ml pipette tip and transferred to 1.5 ml eppendorf tube containing 70% ethanol and centrifuged briefly. The resultant pellet was washed further with two more rounds of 70% ethanol, briefly air-dried, 1 ml of fresh autoclaved water was added and the samples were stored at 4C overnight for the genomic DNA to dissolve. DNA was checked on a 0.7% agarose gel and the total yield was determined to be 100 g each for both PCC4 cells and tissue.